Mass spectrometry with selective ion filtration by digital thresholding

ABSTRACT

The methods described herein generally relate to characterization of large analytes, such as biomolecules, by molecular mass analysis. Specifically, the methods are directed to molecular mass analysis of singly- or multiply-charged ions by selective ion filtering carried out by a digital thresholding process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 60/574,042 filed May 24, 2004, the entiredisclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of detection and characterization oflarge analytes, such as biomolecules, by molecular mass analysis.

BACKGROUND OF THE INVENTION

Mass spectrometry has been used for many decades in the characterizationof small organic molecules. The technique typically involves theionization of molecules in the sample to form molecular ions bysubjecting the sample to an electron beam at a very low pressure. Themolecular ions are then focused and accelerated by an electric fieldinto a magnetic field or quadrupole. The ions are separated in themagnetic field or quadrupole according to the ratio of the mass of theion m to the charge on the ion z (m/z). After passing through the field,the ions impinge upon a detector which determines the intensity of theion beam and the m/z ratio, and these data are used to create the massspectrum of the sample.

With the increasing interest in larger molecules, especiallybiomolecules such as nucleic acids and proteins, new techniques in thefield of mass spectrometry are continually being developed tocharacterize these molecules.

In recent years the performance of commercially available massspectrometers has seen significant improvement due, in part, to theavailability of improved core components including more stable powersupplies, faster digitizers, and more sophisticated fabrication methodsfor ion optic elements. Particularly noteworthy are the newestgeneration ESI-TOF mass spectrometers which, from several vendors in avariety of configurations, are now routinely yielding the types of massmeasurement accuracy and mass resolution previously attainable only onhigh end sector or Fourier transform ion cyclotron resonance(FTICR)-based platforms. As such, the use of such bench top instrumentsby the bioanalytical community continues to expand as these instrumentsare increasingly being made available to scientists and technicians witha broad range of analytical needs. Accordingly, a number of increasinglysophisticated automation schemes are emerging, many incorporating someform of liquid chromatography (LC) as an on-line sample purificationstep to support high throughput QC or drug screening activities. Whilethere are a number of applications in which some form of LC is arequisite step that facilitates the analysis of very complex mixtures,it is also used frequently as a generic desalting/purification protocolto prepare relatively pure analyte fractions for MS analysis.

Low molecular weight chemical noise is often the limiting factor inoverall MS performance as the presence of high levels of low molecularweight components, such as polymers and buffer constituents, candrastically limit the spectral dynamic range and adversely affect massaccuracy. While LC is often used to reduce the adverse affects of suchbackgrounds, constraints on sample throughput and issues associated withsolvent usage/disposal must be considered as part of the laboratory workflow. Additionally, LC is often used as a purification step (as opposedto a separation step) to render analytes amenable to MS analysis.Consequently, there is an increasing need for simple methods to reducethe chemical noise floor and render less than “pristine” samplesamenable to mass spectrometric analysis.

The present invention satisfies this need, as well as others, byproviding systems and methods for digital filtration of mass spectralsignals arising from singly-charged low molecular weight components suchas solution additives and matrix modifiers without significantlyaltering the mass spectral signals of larger analytes such asbiomolecules.

SUMMARY OF THE INVENTION

The present invention is directed to methods of identifying amultiply-charged ion. A mass spectrometer that comprises the followingcomponents is provided: (i) an ion detector, (ii) a digitizer thatconverts an analog signal to a digital signal, (iii) an analog signaltransfer means for transferring an analog signal from the detector tothe digitizer, and (iv) a digital threshold filter which is in digitaldata communication with the digitizer. A digital signal threshold can beset at the digital threshold filter and, in response to a digital signalinput from the digitizer, the digital threshold filter independentlyoutputs a digital signal to a data file only if the digital signal inputis greater than the specified digital signal threshold. The continuingstep of the method is then effected by specifying a digital signalthreshold such that, upon a mass spectrometer measurement of themultiply-charged ion, the filtered digital signal output to the datafile originates from the detection of the multiply-charged ion andexcludes digital signal output from analog signals arising fromsingly-charged ions.

The present invention is also directed to methods for determining themolecular mass of a plurality of analytes in a mixture. A massspectrometer that comprises the following components is provided: (i) anion detector, (ii) a digitizer that converts an analog signal to adigital signal, (iii) an analog signal transfer means for transferringan analog signal from the detector to the digitizer, and (iv) aplurality of digital threshold filters, each in digital datacommunication with the digitizer. A digital signal threshold can beindependently set at any of the plurality of digital threshold filters,each of which is in digital data communication with the digitizer and,in response to a digital signal input from the digitizer, independentlyoutputs a digital signal to a corresponding data file only if thedigital signal input is greater than the specified digital signalthreshold. The continuing steps of the method are then effected byspecifying a unique digital signal threshold at some members of theplurality of digital threshold filters, making a mass spectrometermeasurement of the mixture, wherein each unique digital signal thresholddifferentially filters digital signals arising from the plurality ofanalytes and produces a unique digital signal output to eachcorresponding data file. The measurement results in storage of aplurality of data files. In the final step, each of the plurality ofdata files is analyzed and the molecular mass of at least one member ofthe plurality of analytes is contained in each of the plurality of datafiles.

The present invention is also directed to methods for calibrating a massspectrum of an analyte. A mass spectrometer that comprises the followingcomponents is provided: (i) an ion detector, (ii) a digitizer thatconverts an analog signal to a digital signal, (iii) an analog signaltransfer means for transferring an analog signal from the detector tothe digitizer, and (iv) a plurality of digital threshold filters, eachin digital data communication with the digitizer. A digital signalthreshold can be independently set at any of the plurality of digitalthreshold filters, each of which is in digital data communication withthe digitizer and, in response to a digital signal input from thedigitizer, independently outputs a digital signal to a correspondingdata file only if the digital signal input is greater than the specifieddigital signal threshold. The continuing steps of the method are theneffected by specifying a first unique digital signal threshold at onedigital threshold filter such that digital signal output to a first datafile has signals from both the analyte and a calibrant ion and thenspecifying a second unique digital signal threshold at another digitalthreshold filter such that the digital signal output to a second datafile has signals from the analyte but not the calibrant. The second datafile is subtracted from the first data file to obtain a calibration filewhich is then used to calibrate the mass spectrum.

The present invention is also directed to a system comprised of a massspectrometer that comprises the following components: (i) an iondetector, (ii) a digitizer that converts an analog signal to a digitalsignal, (iii) an analog signal transfer means for transferring an analogsignal from the detector to the digitizer, and (iv) a plurality ofdigital threshold filters for setting a digital signal threshold whichare each in digital data communication with the digitizer and inresponse to a digital signal input from the digitizer independentlyoutputting a digital signal to a corresponding data file only if thedigital signal input is greater than the specified digital signalthreshold. The system has a plurality of data files and a plurality ofparallel digital signal output transferring means, each of which is indigital data communication with one of the plurality of digitalthreshold filters and a corresponding data file from the plurality ofdata files.

BRIEF DISCUSSION OF THE DRAWINGS

FIG. 1 shows the detector response intensity as a function of digitalsignal threshold value (in this case indicated by cutoff voltage) ofanalyte ions having similar m/z ratios but differing in molecularweights. Circles: 140-mer oligonucleotide (m/z=1232.9), squares: 70-meroligonucleotide (m/z=1199), diamonds: 38-mer oligonucleotide(m/z=1174.7), crosses: 12-mer oligonucleotide (m/z=1233) and triangles:polypropylene glycol (PPG−m/z=1236).

FIG. 2 is a schematic representation of the effects of specifyingdigital signal thresholds on mass spectra. FIG. 2 a depicts the rawdigitizer (ADC, analog digital converter) output from a theoreticalsingle scan containing a singly-charged ion (ion1) which strikes thedetector at T1 and a large multiply-charged ion (ion2) which strikes thedetector at T2. FIGS. 2 b and 2 c indicate a spectrum with a high andlow digital signal threshold respectively. FIG. 2 d indicates a spectrumwithout a digital signal threshold and detector “white noise” is visiblein the spectrum.

FIG. 3 displays mass spectra of a PCR product. FIG. 3 a is an ESI-TOFmass spectra of a 140-mer PCR product acquired at a normal (3 mV)digital threshold setting. The sample contains a contaminating amount ofpolypropylene glycol (PPG) relatively high levels of singly chargedpeptides (which serve as internal mass standards). Peaks labeled with“x” indicate signals from the PPG and “c” represents signals from thepeptide mass standards. FIG. 3 b is an ESI-TOF spectrum of the samesample of PCR product obtained at a digital threshold setting of 15 mV.Contaminants and mass standards have been filtered out of the spectrum.

FIG. 4 is an expanded region of the ESI-TOF spectra from FIG. 3 in whichthe relatively low abundance high charge states of the PCR amplicon aredetected. The effective signal to noise of the spectrum in FIG. 4 a isdefined by the signal to chemical noise ratio, while the effectivesignal to noise of the spectrum in FIG. 4 b is defined by the signal toelectronic noise ratio.

FIG. 5 exhibits ESI-TOF spectra of a solution containing approximately0.5 nM PCR product in the presence of 500 nM PPG was characterized atlow (FIG. 5 a) and high (FIG. 5 b) threshold settings As shown in theinset, the top spectrum is also inundated with other chemical noisecomponents and the peak-at-every-mass background precludes the detectionof the low level PCR products. When the digital signal threshold is setsuch that signals from singly charged species are not detected, adistinct signature for the low level amplicon is detected.

FIG. 6 indicates two overlapping peaks of a 140-mer oligonucleotide andof a 12-mer oligonucleotide which can be resolved through acquisition ofdata with different digital signal thresholds. The top spectrum wasobtained with a digital signal threshold setting of 7 mV. The middlespectrum was obtained with a digital signal threshold setting of 11 mV.The bottom spectrum was obtained by subtraction of the middle spectrumfrom the top spectrum to obtain a clean isotopically resolved spectrumof the 12-mer oligonucleotide.

FIG. 7 shows the typical digitizer configuration (FIG. 7 a) with asingle threshold setting compared to a digitizer which allows multiplethreshold settings to be applied simultaneously to data stream comingfrom the TOF digitizer (FIG. 7 b).

FIG. 8 shows mass spectra of carbonic anhydrase in the presence of0.001% SDS and P/I buffer. The protein-derived signals of the spectrumobtained with a 1 mV digital signal threshold setting (FIG. 8 a) aresubject to considerable interference from the detergent and buffercomponents. In contrast, FIG. 8 b indicates that the interferingcomponents are rendered “invisible” by specifying a digital thresholdsetting of 11 mV.

DESCRIPTION OF EMBODIMENTS

In some embodiments of the present invention, the mass spectrometersystem comprises the following components: (i) an ion detector, (ii) adigitizer that converts an analog signal to a digital signal, (iii) ananalog signal transfer means for transferring an analog signal from thedetector to the digitizer, and (iv) a plurality of digital thresholdfilters for setting a digital signal threshold which are each in digitaldata communication with the digitizer and in response to a digitalsignal input from the digitizer independently outputting a digitalsignal to a corresponding data file only if the digital signal input isgreater than the specified digital signal threshold. In someembodiments, the analog and digital signals are voltage signals and theanalog to digital converter (ADC) converts the analog voltage signal toa digital voltage signal. In some embodiments, a plurality of massspectrometer measurements are made and the resulting plurality of datafiles are co-added.

In other embodiments, the mass spectrometer system comprises a (iv)single digital threshold filter instead of a plurality of digitalthreshold filters. The single digital threshold filter is in digitaldata communication with the digitizer and a corresponding data file.

In some embodiments, the mass spectrometer is a time-of-flight massspectrometer, a quadrupole time-of-flight mass spectrometer, a linearquadrupole mass spectrometer, a linear trap mass spectrometer, anelectric/magnetic sector mass spectrometer or a quadrupole ion trap massspectrometer. In some embodiments, ions are produced by electrosprayionization (ESI).

In some embodiments, the multiply-charged analyte is a biomolecule suchas, for example, a nucleic acid, a protein, a carbohydrate or a lipid.Examples of nucleic acids include, but are not limited to, RNAconstructs used to screen small molecules for drug discovery andamplification products such as PCR products which can be used forgenetic analyses. In some embodiments, the multiply-charged analyte isof a molecular weight of 5-500 kDa, 25-250 kDa, or 50-100 kDa.

In some embodiments, the method allows for ESI-TOF characterization ofbiomolecules in the presence of biomolecule stabilizing agents or matrixmodifiers used in online separation techniques. Stabilizing agentsinclude, but are not limited to, buffer salts such as phosphates forexample, ampholytes, glycerol, polyethylene glycol, polypropyleneglycol, reducing agents, detergents, and the like. Matrix modifiers maybe any type of additive used to effect a solution matrix propertyadvantageous to an analytical separation and may include, but are notlimited to, ampholytes, detergents and buffer salts such as phosphatesfor example.

In some embodiments, the biomolecule stabilizing agents or matrixmodifiers are singly-charged when detected by the mass spectrometer. Inother embodiments, the biomolecule stabilizing agents or matrixmodifiers have one or two charges.

In some embodiments, when a plurality of digital signal thresholdfilters are employed in the mass spectrometer system, a plurality ofunique digital signal thresholds are specified in order to obtainparallel differentially filtered data streams which are stored incorresponding data files. In some embodiments, any member of the datafiles may be subtracted from any of the other data files to obtain amore accurate representation of a given analyte signal. Theseembodiments may be used to obtain a more accurate mass spectrum of acalibrant ion, or any other lower molecular weight contaminating ion bysubtracting out an overlapping signal from an ion having a similar m/zbut with a larger molecular mass.

In some embodiments, the methods described herein which employ multipledifferentially thresholded data streams may be used in multiplexed dataacquisition of a plurality of ions such as those obtained from chemical,protease or restriction digestion of proteins or nucleic acids.

In some embodiments, the methods described herein may be used to reducethe burden of level of purification of large molecular weight ormultiply charged analytes such as biomolecules, for example, fromstabilizing agents or matrix modifiers.

EXAMPLES Example 1 ESI-TOF Mass Spectrometry Conditions

A Bruker Daltonics (Billerica, Mass.) MicroTOF ESI time-of-flight (TOF)mass spectrometer was used in this work. Ions from the ESI sourceundergo orthogonal ion extraction and are focused in a reflectron priorto detection. Ions are formed in the standard MicroTOF ESI source whichis equipped with an off-axis sprayer and glass capillary. For operationin the negative ion mode, the atmospheric pressure end of the glasscapillary is biased at 6000 V relative to the ESI needle during dataacquisition. A counter-current flow of dry N2 is employed to assist inthe desolvation process. External ion accumulation is employed toimprove ionization duty cycle during data acquisition. Each ESI-TOFspectrum is comprised of 75,000 data points digitized over 75 μs. Allaspects of data acquisition were controlled by the Bruker MicroTOFsoftware package. Post processing of data was also performed using thestandard Bruker software.

Example 2 PCR Conditions and Purification of Amplification Products

All PCR reactions were assembled in 50 μL reaction volumes in a 96 wellmicrotiter plate format using a Packard MPII liquid handling roboticplatform and M.J. Dyad thermocyclers (MJ research, Waltham, Mass.). ThePCR reaction mix consists of 4 units of Amplitaq Gold, 1× buffer II(Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl₂, 0.4M betaine,800 μM dNTP mix and 250 nM of primer. The following PCR conditions wereused: 95° C. for 10 min followed by 50 cycles of 95° C. for 30 sec, 50°C. for 30 sec, and 72° C. for 30 sec.

PCR products were purified using the protocols disclosed and claimed inU.S. patent application Ser. No. 10/943,344 which is commonly owned andincorporated herein by reference in entirety.

Example 3 Investigation of Detection Efficiency of Large OligonucleotideIons

In an attempt to optimize detection efficiency of large oligonucleotideions, and to better understand the relationship between ion arrivalstatistics and mass accuracy, a detailed systematic study was designedto investigate detector response as a function of molecular weight, m/z,and charge state at the individual ion level.

In time of flight mass spectrometry ions are separated based ondifferences in their velocity as they traverse the flight tube. As ionsstrike the detector, their arrival times are recorded and subsequentlyconverted to m/z based on the specific configuration of the spectrometer(length of flight path, accelerating voltage, geometry, etc.). It isgenerally accepted that for singly charged species, detector response isinversely proportional to molecular weight (velocity) and, for examplein the case of MALDI, higher molecular weight species induce a smallerdetection signal than lower molecular weight species. It was suspectedthat lower charge states (i.e. lower velocity species) induce a smallersignal than do the higher charge states (i.e. high velocity species)under the same accelerating voltages. The reduced response of highmolecular weight “slow” ions can be partially ameliorated by the use ofpost-acceleration methods in which ions are accelerated to very highkinetic energies immediately prior to detection.

During the course of this investigation, it became immediately apparentthat ions of the same nominal m/z but different molecular weightsinduced significantly different detector responses. The heavier, morehighly charged ions consistently produced detector responses severaltimes that of their singly charged counterparts at the same m/z. Thus,while in the TOF mass analyzer ions of the same m/z have the samevelocity, ions of different molecular weigh do not have the samemomentum or kinetic energy and do not induce the same signal on thedetector.

This phenomenon is readily illustrated by examining spectral response asa function of the digital threshold employed to acquire mass spectra ofspecies covering a range of molecular weights.

Unlike MALDI of large biomolecules, the multiple charging phenomenoninherent to the ESI process generally produces mass spectra in which themajority of the signals are in the same m/z range. Molecular ions frommoderate to large biomolecules (1 kDa to 100 kDa) are generally detectedin the 500-2000 m/z range and it is thus not at all uncommon for complexmixtures to yield spectra in which peaks of many different masses aredetected at the same m/z. To characterize detector response as afunction of molecular weight (charge), solutions containing analyteswith molecular weight ratios of 1.0, 3.7, 11.8, 21.5, and 43 wereanalyzed at a range of digital thresholds. For each series, a singlecharge at or near m/z 1233 was used to gauge the detector response. Theresulting molecular weight isopleths are plotted in FIG. 1. Importantly,at low digital signal thresholds set according to Example 4 (videinfra), the singly charged PPG ions drop in intensity at significantlylower cutoff voltages than do the higher molecular weight (charge)species. For example, at a digital signal threshold cutoff voltage of 9mV, the signal of the PPG ions at m/z 1233 is attenuated tonon-detectable levels while the 43 kDa PCR product at m/z 1233 is stilldetected at approximately 90% of the initial response. There is adefinite trend in cutoff voltages as a function of molecular weight(charge state) suggesting that one can select a digital signal thresholdto selectively detect (or not detect) species of interest.

Example 4 Digital Signal Threshold Rationale

Under the acquisition conditions routinely employed to characterize PCRproducts, individual scans are acquired and co-added at a rate of 75kHz. Thus for a typical 45 second acquisition, each spectrum iscomprised of 660,000 co-added individual scans. In order to reduce theshot/white noise in the co-added spectrum, the MicroTOF electronicsallow one to set a digital filter threshold (voltage cutoff) such thatwhite noise from the detector at the single or low-bit ADC count iszeroed out of each scan and only detector responses consistent with iondetection events are passed to the transient summing digitizer datasystem to be co-added. This concept is shown schematically in FIG. 2.FIG. 2 a depicts the raw ADC output from a theoretical single scan inwhich a singly charged ion (ion1) strikes the detector at T1 and a largemultiply charged ion (ion2) which strikes the detector at time T2.During the time intervals in which neither ion1 nor ion2 are strikingthe detector the ADC is picking up and digitizing detector noisegenerally corresponding to 1-5 bits. Because of the fast acquisitionrate of the TOF and the finite ion capacity of the source, each scan istypically comprised of relatively few ion detection events and for anygiven ion channel, it is very unlikely that an ion will be detected ineach scan. Thus, co-adding large numbers of unfiltered scans such asthose depicted in FIG. 2 d would result in a noise floor that increaseslinearly with the number of scans and a mass spectrum in which theultimate dynamic range would be limited by the relatively highelectronic noise floor.

To minimize the deleterious effects of co-adding low-bit detector noise,the MicroTOF electronics allow the user to set a cutoff voltage that hasthe net effect of zeroing-out low level signals that are attributed onlyto detector noise. As illustrated in FIG. 2 c, this approach, ideally,does not affect the ADC counts for signals consistent with a singlycharged ion but digitally filters each scan prior to co-adding, suchthat detector white noise is not co-added with the same efficiency asdetector ion response. As illustrated in FIG. 2 b, this concept can betaken a step further by setting the digital filter threshold such thatADC counts derived from detector noise and singly charged ions strikingthe detector are zeroed out prior to co-adding. Thus, with the digitalthreshold set at the level depicted in FIG. 2 b, a singly charged ionstriking the detector is “invisible” in the post-filtered ADC output andthe net result is a “high pass” molecular weight (charge) filter inwhich low molecular weight (charge) species are not detected but highmolecular weight (charge) species, which tend to be multiply-charged arestill detected.

Example 5 Chemical Noise Removal by High Pass Digital ThresholdFiltering

A key challenge in the analysis of large biopolymers by ESI-MS is samplepurification. Low molecular weight contaminants in biopolymer solutionscan have deleterious effects on the quality of ESI-MS spectra and cansignificantly limit the dynamic range and accuracy of the measurement.In some cases these low molecular weight “contaminants” are actuallyrequired additives as components of an on-line separations. Suchadditives include ampholytes used in capillary isoelectric focusing,phosphates commonly used as components of buffers used in capillary zoneelectrophoresis, and solution matrix modifiers used to promote micelleformation in micellar electrokinetic chromatography. Similarly,electrospray incompatible additives such as glycerol and polymers(polyethelene glycol, PPG) are often used to stabilize enzymes to beused in biochemical processes. These compounds often make their waythrough an entire biochemical process and end up in the massspectrometer. A key example of the latter type of “contaminant” is thepresence of high levels of polyethelene glycol and polypropylene glycolpolymers in the Taq polymerase used for PCR. While typically only 1-2 μLof Taq are used in each 50 μL PCR reaction, the relatively highconcentration of polymer in the presence of the relatively lowconcentration of PCR products (typically 10-100 nM), coupled with thefact that such polymers are ionized with high efficiency, may cause asignificant chemical noise suppression issue.

FIG. 3 a illustrates an example of an ESI-TOF spectrum of a 140-mer PCRproduct into which a contaminating amount of PPG was spiked along withrelatively high levels of singly charged peptides (which serve asinternal mass standards). The signal from the charge state envelope ofthe multiply charged strands of the PCR amplicons is confounded by thepresence of the intense signal arising from the low molecular weightspecies. This spectrum was acquired using a “normal” digital thresholdsetting in which the detector white noise output from the digitizer isfiltered out but the threshold is set low enough to ensure that signalsfrom singly charged ions are captured. This spectrum is exemplary of acommon situation in which a large biopolymer is analyzed in the presenceof a significant chemical noise background arising from low molecularweight contaminants. As shown, such interferences can adversely affectthe mass accuracy of the measurement and result in reduced spectraldynamic range.

In contrast, the ESI-TOF spectrum in FIG. 3 b was acquired on the samespectrometer from the identical solution using the identical ESI sourceparameters and acquisitions conditions with the important exception thatthe spectrum in FIG. 3 b was acquired at a cutoff voltage of 15 mV whilethe spectrum in 3 a was acquired moments earlier at a cutoff voltage of3 mV. It is clear from these spectra, and the data presented in FIG. 2that the 15 mV cutoff setting precludes the detection of the singlycharged species in the solution yet facilitates the detection of thelarger, more highly charged PCR amplicons. It is evident from thespectra in FIG. 3 and the cutoff profiles in FIG. 2 that the intensityof the amplicon peaks are reduced by about 30%; importantly the peaksfrom the singly charged polymer and calibrants are not present in thespectrum acquired at the higher cutoff voltage and the spectrum in FIG.3 b has significantly improved signal-to-chemical noise characteristics.It is worthwhile to emphasize that, no other instrument, solution, ordata processing parameters were changed between collecting the spectrain FIGS. 3 a and 3 b, the only difference was the digital signalthreshold setting.

Indicating the applicability of the method for biomolecules other thannucleic acids, FIG. 8 shows mass spectra of carbonic anhydrase in thepresence of 0.001% SDS and 25 mM Piperidine/Imidizole buffer. Theprotein-derived signals of the spectrum obtained with a 1 mV digitalsignal threshold setting are subject to considerable interference fromthe detergent and buffer components. In contrast, FIG. 8 b indicatesthat the interfering components are rendered “invisible” by specifying adigital threshold setting of 11 mV.

These data indicate that in some high throughput screening and QCapplications a less rigorous sample purification protocol might beemployed and chemical noise can be removed via the digital filteringapproach described above. Importantly, this approach allows ESI-MSanalysis of large biomolecules (or noncovalent complexes) from solutionswhich might otherwise contain too much chemical noise to produceinterpretable spectra.

Example 6 Dynamic Range Enhancement by Digital Threshold Filtering

By reducing or eliminating the chemical noise floor in addition toreducing the electronic noise floor, significant improvements in dynamicrange and spectral quality are attainable. This concept is demonstratedin FIGS. 4 and 5. Shown in FIG. 4 is an expanded region of the ESI-TOFspectra from FIG. 3 in which the relatively low abundance high chargestates of the PCR amplicon are detected. Note that the signals from the(M-43H+)⁴³⁻, (M-42H+)⁴²⁻, and (M-41H+)⁴¹⁻ charge states are barelyvisible in the unfiltered spectrum (FIG. 4 a) but clearly visible in thefiltered spectrum (FIG. 4 b). The effective signal to noise of thespectrum in FIG. 4 a is defined by the signal to chemical noise ratio,while the effective signal to noise of the spectrum in FIG. 4 b isdefined by the signal to electronic noise ratio. For example, for the(M-41H+)⁴¹⁻ charge state of the amplicon the signal to (chemical) noisein the spectrum acquired at the low cutoff threshold is approximately 2while the signal to (electronic) noise of the spectrum acquired at thehigher cutoff threshold is approximately 12. Additionally, signals fromcharge states (M-40H+)⁴⁰⁻ and (M-39H+)³⁹⁻ are not readily discernablefrom the chemical noise in FIG. 4 a but clearly visible in FIG. 4 b.

The improvement in effective dynamic range afforded by the presentinvention is further illustrated in FIG. 5 in which a solutioncontaining approximately 0.5 nM PCR product in the presence of 500 nMPPG was characterized at high and low threshold settings. At the normalthreshold setting the spectrum is dominated by highly abundant singlycharged polymer ions and the very low level PCR products are notobserved. As shown in the inset, the top spectrum is also inundated withother chemical noise components and the peak-at-every-mass backgroundprecludes the detection of the low level PCR products. When the digitalsignal threshold is set such that signals from singly charged speciesare not detected, a distinct signature for the low level amplicon isdetected. This attribute has the potential to significantly improve thedetection of low concentration biomolecules in solution as it isfrequently the presence of low level, ubiquitous, contaminantsintroduced from buffer impurities, plasticware, and sample handling thatdefine the chemical noise floor of the mass spectra and limit theapplicability of ESI-MS to complex biological systems.

In addition to reducing the useful dynamic range of a mass spectrum,chemical noise and low molecular weight contaminants can have adverseaffects on accurate mass measurements. As described above, ESI-MSspectra often have overlapping peaks that result from species ofdifferent molecular weights but the same m/z. This is particularlyproblematic for large biopolymer ions which generally produce somewhatcongested spectra in which multiple charge states are observed in the500 to 2000 m/z range. Because low molecular weight species areisotopically resolved and species above about 10 kDa are generally not,it is quite common to see a low molecular weight contaminant peakoverlap with and distort an otherwise analytically useful analyte peak.An example of this is shown in FIG. 6 in which the signal from the(M-3H+)³⁻ charge state of a 12-mer oligonucleotide is observed at thesame m/z as the (M-35H+)³⁵⁻ charge state of a much larger 140-mer PCRproduct. In this case the smaller oligonucleotide is intended to serveas an internal mass standard but, as is illustrated in FIG. 6 and in themass accuracy data in Table 1, the co-location of these signals isdeleterious to both signals. First, at the 7 mV threshold it is notimmediately apparent that there are two species at m/z 1233 as peaksfrom the isotopically resolved 12-mer mask the presence of the largerunresolved amplicon peak. Additionally, the presence of the unresolvedamplicon peak distorts the peak shapes and centroids of the isotopicallyresolved 12-mer peaks such that the mass accuracy is compromised. Whenthe digital threshold is set to 11 mV, the contribution to the peak fromthe triply charged 12-mer is substantially reduced and the presence of ahigh molecular weight unresolved peak is apparent. Importantly, becausethe aggregate signal (i.e. 12-mer and 140-mer) is captured at the 7 mVdigital threshold level, and the contribution to the signal from the140-mer can be measured at a higher digital threshold level (11 mV inthis example), the signal from the 12-mer can be derived by subtractingthe spectrum acquired at 11 mV from the spectrum acquired at 7 mV. Theresulting spectrum exhibits a notably improved distribution (containing5 peaks) and, perhaps more importantly, the centroided peaks yield areduced mass measurement error across the distribution. In this example,the average mass measurement error for the five peaks was reduced from5.9 to 1.5 ppm following the spectral subtraction.

TABLE 1 Calculated Error in m/z Measurements for 7 mV Digital SignalThreshold vs. (7 mV) − (11 mV) Digital Signal Thresholds Digital SignalPeak Theoretical Measured Threshold (mV) Number (m/z) (m/z) Error (ppm)7 1 1232.5408 1232.5596 −15.2512 7 − 11 1 1232.5408 1232.5449 −3.3246 72 1232.8752 1232.8809 −4.6266 7 − 11 2 1232.8752 1232.8753 −0.0844 7 31233.2095 1233.2005 7.2811 7 − 11 3 1233.2095 1233.2071 1.9292 7 41233.5437 1233.5452 −1.1898 7 − 11 4 1233.5437 1233.5428 0.7558 7 51233.8780 1233.8764 1.2781 7 − 11 5 1233.8780 1233.8799 −1.5585

In Table 1, 7-11 indicates that a spectrum obtained with a digitalsignal threshold setting of 11 mV was subtracted from a spectrumobtained with a digital signal threshold setting of 7 mV.

Example 7 Spectral Subtraction and Ion Partitioning: Obtaining “Slicesof Ions”

In accordance with the present invention, the experiments illustrated inFIGS. 3-5 illustrate that the digital thresholding method describedabove allows for the detection of large multiple charged biomolecularions in such a manner so as to render low molecular weight species“invisible” (based on digital thresholding) while the data presented inFIG. 6 illustrates a method by which low molecular weight species can beanalyzed in such a manner so as to make large multiple chargedbiomolecular ions “invisible” (by digital thresholding and spectralsubtraction). The results from the relatively simple subtractiondescribed in Example 6 lay the foundation for more sophisticated digitalthresholding schemes in which multiple “slices” of a complex ionpopulation can be analyzed simultaneously with the effective resultbeing a multidimensional detection configuration in which ions aresimultaneously measured.

In this work all of the high threshold/low threshold comparisons weremade by multiple measurements of the same analyte solution acquiredunder identical instrument conditions except the digital threshold wasvaried. This was done out of necessity because, as illustrated in FIG. 7a, the basic system architecture of the Bruker MicroTOF consists of asingle data stream from the detector to the digitizer for which a singlethreshold level is applied to the data stream prior to co-adding ofscans. As sample throughput is a key driver in many laboratories,requiring each sample to be analyzed two (or more) times at differentdigital thresholds may not be feasible.

In accordance with the present invention and as a means of circumventingthis problem, the alternative digitization scheme illustrated in FIG. 7b indicates that output from the ADC can be split to multiple paralleldata streams, each of which is subjected to a different digitalthreshold. By subtracting spectra acquired at different digitalthresholds, one could obtain a mass spectrum for any “slice” of the ionpopulation. This would allow one to perform digital thresholding on avery complex mass spectrum and evaluate a range of molecular weights(charges) independent of other, potentially interfering, ion populationssuch as for example, a restriction digest of a nucleic acid or aprotease digest of a protein. Another example could be a biomoleculesuch as a nucleic acid or a protein having a non-convalently-bound smallmolecule.

Having multiple variably-thresholded mass spectra derived from theidentical digitization event would guarantee perfect subtraction ofspectral features and would eliminate potential artifacts which mayarise from spectral drift over the course of acquiring multiple spectra.Importantly, this also means that one could introduce low molecularweight internal mass standards (calibrants) to very accurately calibratethe m/z axis (e.g. the PPG series in FIG. 3 a) but derive accurate massmeasurements of biomolecular analytes from peaks that are never “steppedon” by low molecular weight species (e.g. the digitally thresholdedspectrum in FIG. 3 b).

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, and the like) cited in the present application isincorporated herein by reference in its entirety.

1. A method of identifying a multiply-charged ion comprising: a)specifying a digital signal threshold on a mass spectrometer, whereinthe mass spectrometer comprises: i) an ion detector; ii) a digitizer forconverting an analog signal to a digital signal; iii) an analog signaltransfer means for transferring an analog signal from the detector tothe digitizer; and iv) a digital threshold filter, for setting a digitalsignal threshold, in digital data communication with the digitizer, inresponse to a digital signal input from the digitizer, independentlyoutputting a digital signal to a data file only if the digital signalinput is greater than the specified digital signal threshold; whereinupon a mass spectrometer measurement of the multiply-charged ion, thefiltered digital signal output to the data file originates from thedetection of the multiply-charged ion and excludes digital signal outputfrom analog signals arising from singly-charged ions.
 2. The method ofclaim 1 further comprising making a plurality of mass spectrometermeasurements according to step a) and co-adding the resulting pluralityof data files.
 3. The method of claim 1 wherein the multiply-charged ionis a biomolecule.
 4. The method of claim 3 wherein the biomolecule is anucleic acid, peptide, protein, lipid, or carbohydrate.
 5. The method ofclaim 3 wherein the biomolecule comprises a non-covalently-bound smallmolecule.
 6. The method of claim 1 wherein the singly-charged ions arebiomolecule stabilizer additives or matrix modifiers.
 7. The method ofclaim 6 wherein the stabilizer additives are one or more of:polyethylene glycol, glycerol, reducing agents, detergents or buffersalts, or any combination thereof.
 8. The method of claim 6 wherein thematrix-modifying additives are one or more of: ampholytes, detergents,or buffer salts, or any combination thereof.
 9. The method of claim 1wherein the analog signal is an analog voltage signal and the digitalsignal is a digital voltage signal.
 10. The method of claim 1 whereinthe mass spectrometer is a time-of-flight mass spectrometer, aquadrupole time-of-flight mass spectrometer, a linear quadrupole massspectrometer, a linear trap mass spectrometer, an electric/magneticsector mass spectrometer, or a quadrupole ion trap mass spectrometer.11. A method for determining the molecular mass of a plurality ofanalytes in a mixture comprising: a) specifying a unique digital signalthreshold for at least some members of a plurality of digital thresholdfilters on a mass spectrometer, wherein the mass spectrometer comprises:i) an ion detector; ii) a digitizer for converting an analog signal to adigital signal; iii) an analog signal transfer means for transferring ananalog signal from the detector to the digitizer; and iv) a plurality ofdigital threshold filters for setting a digital signal threshold indigital data communication with the digitizer, in response to a digitalsignal input from the digitizer independently outputting a digitalsignal to a corresponding data file only if the digital signal input isgreater than the specified digital signal threshold; b) making a massspectrometer measurement of the mixture, wherein each unique digitalsignal threshold differentially filters digital signals arising from theplurality of analytes and produces a unique digital signal output toeach corresponding data file wherein the measurement results in storageof a plurality of data files; and c) analyzing each member of theplurality of data files wherein the molecular mass of at least onemember of the plurality of analytes is contained therein.
 12. The methodof claim 11 further comprising making a plurality of mass spectrometermeasurements according to step b) and co-adding the resulting pluralityof data files obtained from the plurality of mass spectrometermeasurements.
 13. The method of claim 11 wherein the analysis of step c)comprises mathematical subtraction of at least one member of theplurality of data files from at least one other member of the pluralityof data files.
 14. The method of claim 11 wherein the plurality ofanalytes comprise singly-charged ions and multiply-charged ions.
 15. Themethod of claim 14 wherein the multiply-charged ions are biomolecules.16. The method of claim 15 wherein the biomolecules comprisenon-covalently-bound small molecules.
 17. The method of claim 15 whereinthe biomolecules are nucleic acids, peptides, proteins, lipids orcarbohydrates.
 18. The method of claim 14 wherein the singly-chargedions are biomolecule stabilizer additives or matrix modifiers.
 19. Themethod of claim 18 wherein the stabilizer additives are one or more of:polyethylene glycol, glycerol, reducing agents, detergents, or buffersalts, or any combination thereof.
 20. The method of claim 18 whereinthe matrix-modifying additives are one or more of: ampholytes,detergents, or buffer salts, or any combination thereof.
 21. The methodof claim 11 wherein the analog signal is an analog voltage signal andthe digital signal is a digital voltage signal.
 22. The method of claim11 wherein the mass spectrometer is a time-of-flight mass spectrometer,a quadrupole time-of-flight mass spectrometer, a linear quadrupole massspectrometer, a linear trap mass spectrometer, an electric/magneticsector mass spectrometer, or a quadrupole ion trap mass spectrometer.23. A method of calibrating a mass spectrum of an analyte comprising: a)specifying a first digital signal threshold at a first member of aplurality of digital threshold filters, on a mass spectrometer, whereinthe mass spectrometer comprises: i) an ion detector; ii) a digitizer forconverting an analog signal to a digital signal; iii) an analog signaltransfer means for transferring an analog signal from the detector tothe digitizer; and iv) a plurality of digital threshold filters forsetting a digital signal threshold, in digital data communication withthe digitizer, in response to a digital signal input from the digitizer,independently outputting a digital signal to a corresponding data fileonly if the digital signal input is greater than the specified digitalsignal threshold; wherein the first digital signal threshold isspecified such that digital signal output to a first data file comprisessignals from the analyte and a calibrant ion; b) specifying a seconddigital signal threshold at a second member of the plurality of digitalthreshold filters wherein the second digital signal threshold isspecified such that digital signal output to a second data filecomprises signals from the analyte but not from the calibrant ion; c)subtracting the second data file from the first data file to obtain acalibration data file comprising a signal from the calibrant ion but notfrom the analyte; and d) using said calibration data file to calibratethe mass spectrum of the analyte.
 24. The method of claim 23 wherein thecalibrant ion is a nucleic acid, peptide or small molecule.
 25. Themethod of claim 23 wherein the analyte is a biomolecule.
 26. The methodof claim 25 wherein the biomolecule is a nucleic acid, peptide, protein,lipid or carbohydrate.
 27. The method of claim 26 wherein thebiomolecule comprises a non-covalently-bound small molecule.
 28. Themethod of claim 23 wherein the analog signal is an analog voltage signaland the digital signal is a digital voltage signal.
 29. The method ofclaim 23 wherein the mass spectrometer is a time-of-flight massspectrometer, a quadrupole time-of-flight mass spectrometer, a linearquadrupole mass spectrometer, a linear trap mass spectrometer, anelectric/magnetic sector mass spectrometer, or a quadrupole ion trapmass spectrometer.
 30. A system comprising a mass spectrometercomprising: an ion detector; a digitizer for converting an analog signalto a digital signal; an analog signal transfer means for transferring ananalog signal from the detector to the digitizer; a plurality of digitalthreshold filters for setting a plurality of independent digital signalthresholds, each digital threshold filter in parallel electroniccommunication with the digitizer, each digital threshold filter inresponse to a digital voltage input from the digitizer independentlyoutputting a digital voltage only if the digital voltage input isgreater than the specified digital signal threshold; a plurality of datafiles; and a plurality of parallel digital signal output transferringmeans, each of which is in digital data communication with one of thedigital threshold filters and a corresponding data file from theplurality of data files.
 31. The system of claim 30 wherein the analogsignal is an analog voltage signal and the digital signal is a digitalvoltage signal.
 32. The system of claim 30 wherein the mass spectrometeris a time-of-flight mass spectrometer, a quadrupole time-of-flight massspectrometer, a linear quadrupole mass spectrometer, a linear trap massspectrometer, an electric/magnetic sector mass spectrometer, or aquadrupole ion trap mass spectrometer.